1. Field of the Invention
The present invention is related to a method of obtaining high purity stem cells from tissue. Particularly, the method of the present invention comprises using a filter device comprising a content to separate impurities from an impurity-containing cell mass and culturing target stem cells on a polymeric film.
2. The Prior Arts
Stem cells are undifferentiated cells capable of self-renewing and multilineage differentiation, which play important roles in a living organism in regard to the development from embryo to mature individual. Even when matured, stem cells still present throughout the living organism and are responsible for the renewing and regeneration of cells among tissues and organs. Stem cells are considered applicable for the treatment of several illnesses such as cancer, Parkinson's disease, Alzheimer's disease, cardiovascular diseases, and immune deficiency syndromes. In recent years, owing to many researches and breakthroughs in the field of stem cell study and biomedical material, stem cells have become one of the important sources of regenerative medicine for the treatment, repair, and rebuilt of damaged human organ tissues.
In terms of functionality, stem cells are multipotent. That is to say, stem cells are able to differentiate into different tissues and organs. Stem cells can be divided into embryonic stem cells and adult stem cells according to their origins. Embryonic stem cells are from the inner cell mass of early embryo sac and remain at the undifferentiated state. Embryonic stem cells are pluripotent stem cells that can differentiate into all cells from the three germ layers of the embryo. Yet, apart from the perfect abilities of renewing and differentiation, the use and obtaining of embryonic stem cells are still of great moral criticism. Adult stem cells, on the other hand, are undifferentiated cells found in mature tissues and are proven obtainable from tissues and organs such as bone marrow, skin, brain, skeletal muscles, and liver. Adult stem cells can self-replicate and differentiate into cells with the functionality of the tissue or organ from which they are originated. The purpose of adult stem cells is to replenish the lost cells under normal metabolism. For adult stem cells, in addition to differentiating into cells with the functionality of the tissue or organ from which they are originated, they can also differentiate into cells with the functionality of other tissues. The use of adult stem cells nowadays lies particularly in the filed of clinical applications since the survival rate of adult stem cells is high and the immunologic rejection is low after transplantation.
The common methods to isolate adult stem cells from tissues utilize density centrifugation to remove tissue fluid and enzymatic digestion followed by extraction to obtain primary cells. Such primary cells are then cultured and selected to give adhesive stem cells. The conventional methods as described above are easily contaminated and require complicate steps of centrifugation and isolation. Although integrated system of sterilized mixer and centrifuge or sealed tubular isolation device for the purpose of decreasing the chance of contamination are available, the purity and yield of stem cells using such system or device are low. In other words, there are still several drawbacks concerning the conventional method of obtaining adult stem cells from tissues such as low purity of the stem cells isolated, risk of contamination and mutation, which are result from contacting with impurities and other heterocytes such as endothelial cells and fibroblasts during the isolation process. These drawbacks largely affect the ability of renewing and differentiation of the stem cells obtained.
Conventionally, immunologic selections are used in order to solve the problem caused by heterocytes, in which flow cytometry or magnetic beads conjugated with antibodies specific to some surface antigens are used to perform stem cell selection. However, the surface antigens of the adult stem cells lack specificity and unity. Furthermore, the adult stem cells from different tissues or different species exhibit different levels of expression of surface antigens. Thus, the selection of adult stem cells via surface antigens cannot effectively cure the defect of the purity of isolated stem cells. Besides, amplifying a large number of cells is required prior to selection using antibodies in order to overcome the lost of cell viability and number caused by the interaction between cells and antibodies during the selection process, which, further increase the duration and cost of the whole isolation and selection process.
For the convenience and applicability of clinical application, a method of obtaining high purity stem cells from tissues is desired and such method should also be able to prevent in vitro contamination of heterocytes or other impurities in the environment that reduces the stemness of the stem cells obtained. Hence, a rapid, simple, and effective method of obtaining high purity stem cells from tissue is of urgent necessity.